The chemotactic response of human dermal fibroblasts to type I, II, and III human collagens and collagenderived peptides was quantitated by an in vitro assay. All three native human collagens and constituent a chains can serve as chemoattractants for fibroblasts in vitro. When type I, II, and III collagens were digested by bacterial collagenase, the resulting peptides were also chemotactic. In addition, synthetic di-and tripeptides containing hydroxyproline were also chemotactic for fibroblasts. Since (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Type IV collagen, isolated initially from basement membranes, has been more difficult than the other three collagens to characterize biochemically and may be heterogeneous (13)(14)(15).We have recently developed a method by which fibroblast chemotaxis to various stimuli can be quantitated in vitro (16). By using this technique, we have isolated and characterized a human lymphocyte-derived chemotactic factor for fibroblasts, LDCF-F (16). In the present study we have found that type I, II, and III collagens and collagen-derived peptides can effect chemotactic migration of fibroblasts. MATERIALS AND METHODS Preparation of Collagens and Constituent a Chains.Human type I and II collagens were obtained from the bone and cartilage of extremities previously amputated to treat peripheral vascular disease. Type III collagen was purified from human livers obtained at autopsy. Collagens were solubilized from these tissues by limited proteolysis with pepsin under nondenaturing conditions and purified by methods previously described (3, 9). Type I collagen was also obtained from the skin of 3-week-old lathyritic chickens (17).Heat-denatured collagen solutions were fractionated on carboxymethyl-cellulose to isolate purified a chains as described (1,3,9).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 871Fibroblast Chemotaxis Assay. Fibroblast chemotaxis was measured in mtro by methods previously described in detail (16). The assay uses blindwell modified Boyden chemotaxis chambers equipped with gelatin-treated polycarbonate filters having 8-,m pores. Normal human dermal fibroblasts maintained as continuous cell lines were used as indicator cells in the assay. Fibroblasts were harvested from monolayers by either trypsinization or EDTA treatment. Fibroblasts chemotactic activity was determined after chambers were incubated for 150 min by counting the fibroblasts migrating to the lower surface of the polycarbonate filters in 20 oil immersion fields. Substances were assayed in triplicate, and the final chemotactic activity of a sample was expressed as the mean + SEM.Lyophilized collagen and a chain preparations were solubilized for use in the chemotaxis studies by dissolving them in 0.5 M acetic acid, stirring them overnight (40), and then dialyzing them against large volumes of 10 mM phosphate/0.14 M NaCl at p...

The skin has developed a hierarchy of systems that encompasses the skin immune and local steroidogenic activities in order to protect the body against the external environment and biological factors and to maintain local homeostasis. Most recently it has been established that skin cells contain the entire biochemical apparatus necessary for production of glucocorticoids, androgens and estrogens either from precursors of systemic origin or, alternatively, through the conversion of cholesterol to pregnenolone and its subsequent transformation to biologically active steroids. Examples of these products are corticosterone, cortisol, testosterone, dihydrotesterone and estradiol. Their local production can be regulated by locally produced corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH) or cytokines. Furthermore the production of glucocorticoids is affected by ultraviolet B radiation. The level of production and nature of the final steroid products are dependent on the cell type or cutaneous compartment, e.g., epidermis, dermis, adnexal structures or adipose tissue. Locally produced glucocorticoids, androgens and estrogens affect functions of the epidermis and adnexal structures as well as local immune activity. Malfunction of these steroidogenic activities can lead to inflammatory disorders or autoimmune diseases. The cutaneous steroidogenic system can also have systemic effects, which are emphasized by significant skin contribution to circulating androgens and/or estrogens. Furthermore, local activity of CYP11A1 can produce novel 7 -steroids and secosteroids that are biologically active. Therefore, modulation of local steroidogenic activity may serve as a new therapeutic approach for treatment of inflammatory disorders, autoimmune processes or other skin disorders. In conclusion, the skin can be defined as an independent steroidogenic organ, whose activity can affect its functions and the development of local or systemic inflammatory or autoimmune diseases.

Transforming growth factor beta (TGF-beta) is a potent chemoattractant in vitro for human dermal fibroblasts. Intact disulfide and perhaps the dimeric structure of TGF-beta is essential for its ability to stimulate chemotactic migration of fibroblasts, since reduction with 2-ME results in a marked loss of its potency as a chemoattractant. Although epidermal growth factor (EGF) appears to be capable of modulating some effects of TGF-beta, it does not alter the chemotactic response of fibroblasts to TGF-beta. Specific polyvalent rabbit antibodies to homogeneously pure TGF-beta block its chemotactic activity but has no effect on the other chemoattractants tested (platelet-derived growth factor, fibronectin, and denatured type I collagen). Since TGF-beta is secreted by a variety of neoplastic and normal cells including platelets, monocytes/macrophages, and lymphocytes, it may play a critical role in vivo in embryogenesis, host response to tumors, and the repair response that follows damage to tissues by immune and nonimmune reactions.

We define previously unrecognized in vivo pathways of vitamin D(3) (D3) metabolism generating novel D3-hydroxyderivatives different from 25-hydroxyvitamin D(3) [25(OH)D3] and 1,25(OH)(2)D3. Their novel products include 20-hydroxyvitamin D(3) [20(OH)D3], 22(OH)D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3, 1,20(OH)(2)D3, 1,20,23(OH)(3)D3, and 17,20,23(OH)(3)D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ∼20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α-hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH)(2)D3 → 17,20,23(OH)(3)D3 with further 1α-hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH)(2)D3 and D3 → 22(OH)D3 → 20,22(OH)(2)D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH)(2)D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3, and 17,20,23(OH)(3)D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH)(2)D3.

RORα and RORγ are expressed in human skin cells that produce the noncalcemic 20-hydroxyvitamin D3 [20(OH)D3] and 20,23-dihydroxyvitamin D3 [20,23(OH)2D3]. Chinese hamster ovary (CHO) cells stably expressing a Tet-on RORα or RORγ expression vector and a ROR-responsive element (RORE)-LUC reporter, and a mammalian 2-hybrid model examining the interaction between the ligand binding domain (LBD) of RORα or RORγ with an LBD-interacting LXXLL-peptide, were used to study ROR-antagonist activities. These assays revealed that 20(OH)D3 and 20,23(OH)2D3 function as antagonists of RORα and RORγ. Moreover, 20(OH)D3 inhibited the activation of the promoter of the Bmal1 and G6pase genes, targets of RORα, and 20(OH)D3 and 20,23(OH)2D3 inhibited Il17 promoter activity in Jurkat cells overexpressing RORα or RORγ. Molecular modeling using crystal structures of the LBDs of RORα and RORγ revealed docking scores for 20(OH)D3, 20,23(OH)2D3 and 1,25(OH)2D3 similar to those of the natural ligands, predicting good binding to the receptor. Notably, 20(OH)D3, 20,23(OH)2D3, and 1,25(OH)2D3 inhibited RORE-mediated activation of a reporter in keratinocytes and melanoma cells and inhibited IL-17 production by immune cells. Our study identifies a novel signaling pathway, in which 20(OH)D3 and 20,23(OH)2D3 act as antagonists or inverse agonists of RORα and RORγ, that opens new possibilities for local (skin) or systemic regulation.-Slominski, A. T., Kim, T.-K., Takeda, Y., Janjetovic, Z., Broz˙yna, A. A., Skobowiat, C., Wang, J., Postlethwaite, A., Li, W., Tuckey, R. C., Jetten, A. M. RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D.

Systemic scleroderma (SSc) is one of the most complex systemic autoimmune diseases. It targets the vasculature, connective tissue-producing cells (namely fibroblasts/myofibroblasts), and components of the innate and adaptive immune systems. Clinical and pathologic manifestations of SSc are the result of: (1) innate/adaptive immune system abnormalities leading to production of autoantibodies and cell-mediated autoimmunity, (2) microvascular endothelial cell/small vessel fibroproliferative vasculopathy, and (3) fibroblast dysfunction generating excessive accumulation of collagen and other matrix components in skin and internal organs. All three of these processes interact and affect each other. The disease is heterogeneous in its clinical presentation that likely reflects different genetic or triggering factor (i.e., infection or environmental toxin) influences on the immune system, vasculature, and connective tissue cells. The roles played by other ubiquitous molecular entities (such as lysophospholipids, endocannabinoids, and their diverse receptors and vitamin D) in influencing the immune system, vasculature, and connective tissue cells are just beginning to be realized and studied and may provide insights into new therapeutic approaches to treat SSc.

Research over the last decade has revealed that CYP11A1 can hydroxylate the side chain of vitamin D3 at carbons 17, 20, 22 and 23 to produce at least 10 metabolites, with 20(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3, 17,20(OH)2D3 and 17,20,23(OH)3D3 being the main products. However, CYP11A1 does not act on 25(OH)D3. The placenta, adrenal glands and epidermal keratinocytes have been shown to metabolize vitamin D3 via this CYP11A1-mediated pathway that is modified by the activity of CYP27B1, with 20(OH)D3 (the major metabolite), 20,23(OH)2D3, 1,20(OH)2D3, 1,20,23(OH)3D3 and 17,20,23(OH)3D3 being detected, defining these secosteroids as endogenous regulators/natural products. This is supported by the detection of a mono-hydroxyvitamin D3 with the retention time of 20(OH)D3 in human serum. In new work presented here we demonstrate that the CYP11A1-initiated pathways also occurs in Caco-2 colon cells. Our previous studies show that 20(OH)D3 and 20,23(OH)2D3 are non-calcemic at pharmacological doses, dependent in part on their lack of a C1α hydroxyl group. In epidermal keratinocytes, 20(OH)D3, 20(OH)D2 and 20,23(OH)2D3 inhibited cell proliferation, stimulated differentiation and inhibited NF-κB activity with potencies comparable to 1,25(OH)2D3, acting as partial agonists on the VDR. 22(OH)D3 and 20,22(OH)2D3, as well as secosteroids with a short or no side chain, showed antiproliferative and prodifferentiation effects, however, with lower potency than 20(OH)D3 and 20,23(OH)2D3. The CYP11A1-derived secosteroids also inhibited melanocyte proliferation while having no effect on melano-genesis, and showed anti-melanoma activities in terms of inhibiting proliferation and the ability to grow in soft agar. Furthermore, 20(OH)D3 and 20,23(OH)2D3 showed anti-fibrosing effects in vitro, and also in vivo for the former. New data presented here shows that 20(OH)D3 inhibits LPS-induced production of TNFα in the J774 line, TNFα and IL-6 in peritoneal macrophages and suppresses the production of proinflammatory Th1/Th17-related cytokines, while promoting the production of the anti-inflammatory cytokine IL-10 in vivo. In summary, CYP11A1 initiates new pathways of vitamin D metabolism in a range of tissues and products could have important physiological roles at the local or systemic level. In the skin, CYP11A1-derived secosteroids could serve both as endogenous regulators of skin functions and as excellent candidates for treatment of hyperproliferative and inflammatory skin disorders, and skin cancer. This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’.

Interleukin 4 (also known as "B cell stimulatory factor-l"), a cytokine product of T lymphocytes and mast cells, stimulates synthesis of the extracellular matrix proteins, types I and III collagen and fibronectin, by human dermal fibroblasts in vitro. Stimulation of collagen by human recombinant (hr)IL-4 was also demonstrated in several fibroblastic synovial cell lines obtained from patients with rheumatoid arthritis and osteoarthritis. The stimulatory effect of hrIL-4 on fibroblast collagen synthesis was specifically neutralized by rabbit anti-hrIL-4 Ig. IL-4 specifically increased the steady-state levels of types I and III procollagen and fibronectin mRNAs, with no effect on cytoplasmic fl-actin mRNA. Quantitative analysis of the levels of Pro al (I) collagen transcripts in IL-4-treated fibroblast cultures was also corroborated by antisense RNA-mRNA hybridization and RNAse resistant hybrids which showed that IL-4-treated fibroblasts expressed higher levels of Pro al (I) collagen transcripts. Nuclear run-off transcription experiments indicated that IL4 stimulated the rates of mRNA biogenesis. Based on these observations we conclude that IL4 exerts its effect on collagen and fibronectin synthesis at the pretranslational level, resulting in synthesis of these extracellular matrix proteins. These and other data suggest that IL4 may be a "fibrogenic cytokine" that could be important in promoting biogenesis of extracellular matrix proteins in normal wound healing and in pathological fibrosis in which mast cells and T lymphocytes play a central role. (J. Clin. Invest. 1992.